1. Field of the Invention
This invention relates generally to nucleic acid sequences encoding polypeptides that are associated with increased growth in plants. In particular, this invention relates to nucleic acid sequences encoding polypeptides that confer upon a plant increased growth under normal or abiotic stress conditions and/or increased tolerance under abiotic stress conditions.
2. Background Art
Abiotic environmental stresses, such as drought stress, salinity stress, heat stress, and cold stress, are major limiting factors of plant growth and productivity. Crop losses and crop yield losses of major crops, such as soybean, rice, maize (corn), cotton, and wheat caused by these stresses represent a significant economic and political factor and contribute to food shortages in many underdeveloped countries.
Plants are typically exposed during their life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against these conditions of desiccation. However, if the severity and duration of the drought conditions are too great, the effects on development, growth, and yield of most crop plants are profound. Continuous exposure to drought conditions causes major alterations in the plant metabolism, which ultimately lead to cell death and consequently yield losses.
Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems. However, traditional plant breeding strategies to develop new lines of plants that exhibit resistance (tolerance) to these types of stresses are relatively slow and require specific resistant lines for crossing with the desired line. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Additionally, the cellular processes leading to drought, cold, and salt tolerance in model drought-, cold-, and/or salt-tolerant plants are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways. This multi-component nature of stress tolerance has not only made breeding for tolerance largely unsuccessful, but also has limited the ability to genetically engineer stress tolerant plants using biotechnological methods.
Drought stresses, heat stresses, cold stresses, and salt stresses, have a common theme important for plant growth, and that is water availability. As discussed above, most plants have evolved strategies to protect themselves against these conditions of desiccation, however, if the severity and duration of the drought conditions are too great, the effects on plant development, growth, and yield of most crop plants are profound. Furthermore, most of the crop plants are very susceptible to higher salt concentrations in the soil. Because high salt content in some soils results in less water being available for cell intake, high salt concentration has an effect on plants similar to the effect of drought on plants. Additionally, under freezing temperatures, plant cells lose water as a result of ice formation that starts in the apoplast and withdraws water from the symplast. A plant's molecular response mechanisms to each of these stress conditions are common, and ion transporters play an essential role in these molecular mechanisms.
Common damage from different stresses such as drought, salinity, and cold stress, appears to be mostly due to dehydration (Smirnoff, 1998, Curr. Opin. Biotech. 9:214-219). Drought (water stress)-tolerant and -sensitive plants can be clearly distinguished by the dramatic accumulation of ions and solutes in tolerant plants that leads to osmotic adjustments (Bohnert and Jensen, 1996, TIBTECH 14:89-97). Drought and high salt conditions may interact with mineral nutrition in a number of ways as a consequence of (1) reduced transport of ions through the soil to the roots; and/or (2) modified uptake of ions by the roots.
Potassium is a major plant macronutrient, and the potassium cation is the most abundant cation in plants. Movement and transport of potassium is important for plant growth, development, osmoregulation, and homeostasis. Potassium transporters in plants have been identified as belonging to several families, including the HAK family. The HAK family contains members with homology to potassium transporters first identified in Escherichia coli and Schwanniomyces occidentalis. These HAK potassium transporters are found as large gene families in Arabidopsis (Maeser et al., 2001) and in rice (Banuelos et al., 2002). Hydrophobicity profiles of this HAK class of potassium transporters suggest that these proteins possess 12 transmembrance domains and a long cytosolic loop. Some members of this family have been demonstrated to function as low-affinity potassium cation transporters (Quintero et al., 1997) while in other organisms the potassium cation transporter has been demonstrated to function as a high-affinity transporter (Rubio et al., 2000).
Potassium is particularly important in plants not only as a nutrient, but also as an osmoticum. Potassium can make a 30-50% contribution to water potential, particularly in older leaf tissues (Munns et al., 1979, Aust. J. Plant Physiol. 6:379-389). After prolonged drought in the field, potassium accumulates in leaves of ryegrass and barley, and could have a role in osmotic adjustment. In addition, potassium plays a key role in the opening of the stomata. Because potassium is lost from the guard cells (Ehret and Boyer, 1979, J. Exper. Bot. 30:225-234), a reduced supply of potassium reduces stomatal conductance to CO2 much more than it reduces internal conductance (Terry and Ulrich, 1973, Plant Physiol. 51:783-786).
Plant roots can absorb potassium over more than a 1000-fold concentration range, and the concentration dependence of potassium uptake by roots has complex kinetics, suggesting the presence of multiple potassium uptake systems. Gene families encoding inward-rectifying K+ channels have been identified in several plant species. The AKT1 K+ channel gene is predominantly expressed in roots and genetic analysis indicates that the AKT1 channel mediates the uptake of K+ in both the micromolar and millimolar ranges (Hirsch et al., 1998, Science 280:918-921). Active transporters also participate in K+ uptake, and several candidate genes encoding energized transporters have been identified (Hirsch and Sussman, 1999, TIBTECH 17:356-361).
Plant biomass is yield for forage crops like alfalfa, silage corn, and hay. Many proxies for yield have been used in grain crops. Chief amongst these are estimates of plant size. Plant size can be measured in many ways depending on species and developmental stage, but include total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number and leaf number. Many species maintain a conservative ratio between the size of different parts of the plant at a given developmental stage. These allometric relationships are used to extrapolate from one of these measures of size to another (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105: 213). Plant size at an early developmental stage will typically correlate with plant size later in development. A larger plant with a greater leaf area can typically absorb more light and carbon dioxide than a smaller plant and therefore will likely gain a greater weight during the same period (Fasoula & Tollenaar 2005 Maydica 50:39). This is in addition to the potential continuation of the micro-environmental or genetic advantage that the plant had to achieve the larger size initially. There is a strong genetic component to plant size and growth rate (e.g. ter Steege et al 2005 Plant Physiology 139:1078), and so for a range of diverse genotypes plant size under one environmental condition is likely to correlate with size under another (Hittalmani et al 2003 Theoretical Applied Genetics 107:679). In this way a standard environment is used as a proxy for the diverse and dynamic environments encountered at different locations and times by crops in the field.
Harvest index, the ratio of seed yield to above-ground dry weight, is relatively stable under many environmental conditions and so a robust correlation between plant size and grain yield can often be obtained (e.g. Rebetzke et al 2002 Crop Science 42:739). These processes are intrinsically linked because the majority of grain biomass is dependent on current or stored photosynthetic productivity by the leaves and stem of the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa State University Press, pp 68-73) Therefore, selecting for plant size, even at early stages of development, has been used as an indicator for future potential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105: 213). When testing for the impact of genetic differences on stress tolerance, the ability to standardize soil properties, temperature, water, and nutrient availability and light intensity is an intrinsic advantage of greenhouse or plant growth chamber environments compared to the field. However, artificial limitations on yield due to poor pollination due to the absence of wind or insects, or insufficient space for mature root or canopy growth, can restrict the use of these controlled environments for testing yield differences. Therefore, measurements of plant size in early development, under standardized conditions in a growth chamber or greenhouse, are standard practices to provide indication of potential genetic yield advantages.
There is a fundamental physiochemically-constrained trade-off, in all terrestrial, photosynthetic organisms, between carbon dioxide (CO2) absorption and water loss (Taiz and Zeiger, 1991, Plant Physiology, Benjamin/Cummings Publishing Co., p. 94). CO2 needs to be in aqueous solution for the action of CO2 fixation enzymes such as Rubisco (Ribulose 1,5-bisphosphate Carboxylase/Oxygenase) and PEPC (Phosphoenolpyruvate carboxylase). As a wet cell surface is required for CO2 diffusion, evaporation will inevitably occur when the humidity is below 100% (Taiz and Zeiger, 1991, p. 257). Plants have numerous physiological mechanisms to reduce water loss (e.g. waxy cuticles, stomatal closure, leaf hairs, sunken stomatal pits). As these barriers do not discriminate between water and CO2 flux, these water conservation measures will also act to increase resistance to CO2 uptake (Kramer, 1983, Water Relations of Plants, Academic Press p. 305). Photosynthetic CO2 uptake is absolutely required for plant growth and biomass accumulation in photoautotrophic plants.
Water Use Efficiency (WUE) is a parameter frequently used to estimate the trade off between water consumption and CO2 uptake/growth (Kramer, 1983, Water Relations of Plants, Academic Press p. 405). WUE has been defined and measured in multiple ways. One approach is to calculate the ratio of whole plant dry weight, to the weight of water consumed by the plant throughout its life (Chu et al., 1992, Oecologia 89:580). Another variation is to use a shorter time interval when biomass accumulation and water use are measured (Mian et al., 1998, Crop Sci. 38:390). Another approach is to utilize measurements from restricted parts of the plant, for example, measuring only aerial growth and water use (Nienhuis et al 1994 Amer J Bot 81:943). WUE also has been defined as the ratio of CO2 uptake to water vapor loss from a leaf or portion of a leaf, often measured over a very short time period (e.g. seconds/minutes) (Kramer, 1983, p. 406). The ratio of 13C/12C fixed in plant tissue, and measured with an isotope ratio mass-spectrometer, also has been used to estimate WUE in plants using C3 photosynthesis (Martin et al., 1999, Crop Sci. 1775).
An increase in WUE is informative about the relatively improved efficiency of growth and water consumption, but this information taken alone does not indicate whether one of these two processes has changed or both have changed. In selecting traits for improving crops, an increase in WUE due to a decrease in water use, without a change in growth would have particular merit in an irrigated agricultural system where the water input costs were high. An increase in WUE driven mainly by an increase in growth without a corresponding jump in water use would have applicability to all agricultural systems. In many agricultural systems where water supply is not limiting, an increase in growth, even if it came at the expense of an increased water use (i.e. no change in WUE), could also increase yield. Therefore new methods to increase both WUE and biomass accumulation are required to improve agricultural productivity. As WUE integrates many physiological processes relating to primary metabolism and water use, it is typically a highly polygenic trait with a large genotype by environment interaction (Richards et al., 2002, Crop Sci. 42:111). For these and other reasons, few attempts to select for WUE changes in traditional breeding programs have been successful.
Although some genes that are involved in stress responses and water use efficiency in plants have been characterized, the characterization and cloning of plant genes that confer stress tolerance and water use efficiency remains largely incomplete and fragmented. For example, certain studies have indicated that drought and salt stress in some plants may be due to additive gene effects, in contrast to other research that indicates specific genes are transcriptionally activated in vegetative tissue of plants under osmotic stress conditions. Although it is generally assumed that stress-induced proteins have a role in tolerance, direct evidence is still lacking, and the functions of many stress-responsive genes are unknown.
There is a need, therefore, to identify additional genes expressed in stress tolerant plants and plants that are efficient in water use that have the capacity to confer stress tolerance and/or increased water use efficiency to the host plant and to other plant species. Newly generated stress tolerant plants and plants with increased water use efficiency will have many advantages, such as an increased range in which the crop plants can be cultivated, by for example, decreasing the water requirements of a plant species. Other desirable advantages include increased resistance to lodging, the bending of shoots or stems in response to wind, rain, pests, or disease.